The proposed research aims to elucidate the cellular-molecular mechanisms in the mammalian retina that continuously rejuvenate the light-sensitive outer segment portions of photoreceptor neurons. This fundamental retinal process, known as outer segment renewal, is essential for life-long visual function: Experimental deletion of any gene known thus far to encode a protein of the conserved mammalian outer segment renewal machinery results in progressive loss of photoreceptor function and, eventually, photoreceptor degeneration in rodent models. Furthermore, inherited aberration in outer segment renewal causes forms of retinitis pigmentosa in human patients that are characterized by severe retinal degeneration. Finally, inefficiency of outer segment renewal with age causes lipofuscin accumulation in the RPE in the human eye, which impairs a number of RPE functions contributing to dysfunction associated with age-related macular degeneration. Mammalian outer segment renewal is a synchronized circadian process that involves collaboration of both photoreceptors and neighboring retinal pigment epithelial (RPE) cells. Diurnal exposure of the anionic membrane lipid phosphatidylserine (PS) at distal tips of rod outer segments triggers their shedding and simultaneous clearance phagocytosis by RPE cells. To date, PS exposure is the only confirmed molecular eat-me signal of spent cells (e.g. of any cell undergoing apoptosis) and of shed outer segment tips. Despite its universality, the molecular mechanisms triggering PS exposure are still largely obscure in any cell type. We propose two complementary but independent specific aims to identify molecules, activities and their functional interactions that control decorating distal rod outer segment tips with PS in a circadia rhythm in the mammalian retina. Employing innovative live and fixed imaging and quantitative biochemical approaches will allow tracking the entire outer segment renewal process in mutant animals that lack candidate proteins we hypothesize to govern rod PS exposure. In specific aim 1, focus will be on the role of the transmembrane phospholipid scramblase TMEM16F, which localizes to rod outer segment plasma membranes where we propose it to control membrane asymmetry to yield diurnal PS-marked tips. In support, we show: -TMEM16F is elevated in the retina at light onset; -inhibition of TMEM16F prevents ex vivo triggering of PS exposure at rod tips; -mice lacking TMEM16F phenocopy RCS rats and MerTK-/- mice defective in RPE phagocytosis, with normal outer segment development but abnormal RPE phagocytosis followed by progressive photoreceptor loss. In specific aim 2, focus will be on the role of the secreted carbohydrate binding protein galectin-1, which resides in the subretinal space where we propose it to contribute to PS exposure at rod outer segment tips from the extracellular side. In support, we show: -RPE cells are a source of galectin-1; -the PS-externalizing form of galectin-1 is elevated in the retina at light onset; -adding galectin-1 triggers PS externalizationat rod tips; -outer segment renewal in mice lacking galectin-1 is abnormal.